Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include features (sometimes referenced as spots or regions) of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. The array is “addressable” in that different features have different predetermined locations (“addresses”) on a substrate carrying the array.
Biopolymer arrays can be fabricated using in situ synthesis methods or deposition of the previously obtained biopolymers. The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA). In situ methods also include photolithographic techniques such as described, for example, in WO 91/07087, WO 92/10587, WO 92/10588, and U.S. Pat. No. 5,143,854. The deposition methods basically involve depositing biopolymers at predetermined locations on a substrate which are suitably activated such that the biopolymers can link thereto. Biopolymers of different sequence may be deposited at different feature locations on the substrate to yield the completed array. Washing or other additional steps may also be used. Procedures known in the art for deposition of polynucleotides, particularly DNA such as whole oligomers or cDNA, are described, for example, in U.S. Pat. No. 5,807,522 (touching drop dispensers to a substrate), and in PCT publications WO 95/25116 and WO 98/41531, and elsewhere (use of an ink jet type head to fire drops onto the substrate).
In array fabrication, the quantities of DNA available for the array are usually very small and expensive. Sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require the manufacture and use of arrays with large numbers of very small, closely spaced features.
The arrays, when exposed to a sample, will exhibit a binding pattern. The array can be interrogated by observing this binding pattern by, for example, labeling all polynucleotide targets (for example, DNA) in the sample with a suitable label (such as a fluorescent compound), scanning an interrogating light across the array and accurately observing the fluorescent light (sometimes referenced as a “light signal” or “signal”) from the different features of the array. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample. Peptide arrays can be used in a similar manner. Techniques for scanning arrays are described, for example, in U.S. Pat. No. 5,763,870 and U.S. Pat. No. 5,945,679. However, the light detected from respective features emitted in response to the interrogating light, may be other than fluorescence from a fluorescent label. For example, the detected light may be from fluorescence polarization, reflectance, or scattering, as described in U.S. Pat. No. 5,721,435.
Array scanners typically use a laser as an interrogating light source, which is scanned over the array features. Particularly in array scanners used for DNA sequencing or gene expression studies, a detector (typically a fluorescence detector) with a very high light sensitivity is normally desirable to achieve maximum signal-to-noise in detecting hybridized molecules. At present, photomultiplier tubes (“PMTs”) are still the detectors of choice although charge coupled devices (“CCDs”) can also be used. PMTs are typically used for temporally sequential scanning of array features, while CCDs permit scanning many features in parallel. Often a confocal detector system is used which to provide high depth discrimination and thereby reduce noise such as fluorescence of the substrate. However, this also results in capture of only a very small proportion of the emitted fluorescent light.
While detectors may be highly sensitive, the fluorescence detected may still be very weak, particularly where very little of a fluorescently labeled target is bound to a particular array feature. Weak signals may lead to errors in array interrogation and subsequent misinterpretation of results. Interrogating light power to a feature can be increased but this requires a more powerful source (typically a laser). Furthermore, increasing interrogating light power to a feature is not always an option, since fluorescent moieties rapidly become saturated such that an increase in interrogating light power does not increase signal, but may increase noise.
The present invention realizes that it would be desirable then, when possible without saturation, to provide a high interrogation light power at a feature without necessarily having to increase the output available from the interrogating light source. The present invention further realizes that it would be desirable to detect as much of the signal emitted from a feature as possible, without having to further increase interrogating light power, while maintaining detected noise at a low level.